Methionine residues in proteins can be oxidized by a variety of oxidants, such as hydrogen peroxide or peroxynitrite. While oxidation to methionine sulfone is irreversible, methionine sulfoxide can be enzymatically reduced back to methionine by methionine sulfoxide reductase. The role of methionine oxidation in protein is still poorly understood, but it has been suggested that methionine oxidation/reduction works as an oxidant scavenger system, protecting proteins from further deleterious damage. There is, however, increasing evidence that methionine oxidation/reduction might also be involved in gene expression modulation and in regulation of signal transduction and enzymatic activity. This form of post-translational protein modification has received little attention chiefly due to the lack of a sensitive analytical method for the detection of methionine sulfoxides in intact proteins. We have therefore sought to develop such a method by using known chemical reactions to selectively modify methionine sulfoxides to functional groups more readily detectable. In one approach, alkyl sulfoxides bearing alpha protons are treated with an acid anhydride, such as trifluoroacetic anhydride, to form alpha-acyloxysulfides. In the specific case of methionine sulfoxide, the expected product is epsilon-acyloxy methionine. Acid hydrolysis of this product releases homocysteine, which can be conveniently assayed with sulfhydryl specific reagents. We have found that free methionine sulfoxide is quantitatively converted to homocysteine when treated in trifluoroacetic anhydride at 37C for 40 min and hydrolyzed in dilute acid. However, under identical conditions, the methyl esters of N-benzoyl methionine sulfoxide and N-acetyl methionine sulfoxide (models of methionine sulfoxide in proteins) did not yield the desired products. MS and NMR analysis of the reaction mixtures obtained prior to hydrolysis revealed the rapid formation of a stable five-membered cyclic azasulfonium salt. In the presence of trace amounts of water, this intermediate reverts to the sulfoxide. However, it was converted to the alpha-trifluoroacetyloxy sulfide by heating in a non-polar solvent, such as hexane or by treating with triethylamine. Subsequent acid hydrolysis gave the N-acyl homocysteine methyl esters in almost quantitative yield.We are currently testing this method with small methionine sulfoxide-containing peptides and proteins.